1. Announcements Volunteer note taker Tutorial next Thursday

2. Today 1.Action Potential 2.Ionic Basis of Action Potential

3. Action Potentials What are they? –Rapid reversal of the resting membrane potential cell 0 mV -80 mV 3 ms

4. Electrophysiology Techniques Lolligo pealeii Squid giant axon -1 mm in diameter -1000X larger than most

5. axon Recording electrode Reference electrode stimulus

6. Action Potentials Important concepts: Threshold All or none Regenerative Conduction along axons

7. Action Potential 0 mV -80 mV Rising phase or depolarization Falling phase or Repolarization Resting membrane potential Threshold Potential Undershoot or after- hyperpolarization Overshoot

8. Reminders… 1.Action potential is a rapid reversal of Vm 2.Vm is dominated by the equilibrium potential of the most permeable ion 3.Permeability controlled by ion channels

9. Ionic Basis of Action Potentials 0 mV -80 mV P K >>P Na P Na >>P K P K >>P Na E Na EKEK Time 

10. K+ Na+ Voltage-gated channels K+ leak channel Na+ K+ Section of Squid Axon Membrane Potential time

11. K+ Na+ K+ time Membrane Potential

12. 1.At rest only K+ leak channels open, P K >>P Na 2.With stimulus, voltage-gated Na channels open, P Na >>P K Na+ flows into the cell carrying positive charge 3.Delayed opening of voltage-gated K channels, P K >>P Na K+ flows out of cell removing positive charge

13. K+ Na+ K+

14. How do we know Na+ important for depolarization? 0 mV -80 mV Replace Na+ in extracellular bath with impermeable cation - choline Normal Low Sodium

15. Ion currents underlying the AP Use voltage-clamp technique to measure currents Measure currents in the presence and absence of Na+

16. What are ionic currents? –So far, voltage (V) –When ions move  current (I) –Movement through channel is resistance (R) Reciprocal is conductance (g) Ohm’s law I=V/R or I=gV Where g = 1 / R

17. More properly I ion = g ion X emf ion I ion is ionic current g ion is ionic conductance emf ion is the electromotive force acting on an ion emf ion = Vm - E ion Total membrane potential Nernst potential for the ion

18. Therefore, g ion is controlled by ion channels If all channels closed, g = 0 and no ions flow if Vm = E ion then emf = 0, and no ions flow

19. How are ion currents measured? Voltage-clamp

20. Electrophysiology Techniques Voltage clamp Squid axon Recording electrode Reference electrode Command Signal ImIm Current output Control amplifier Voltage output +- Current electrodes Membrane currents

21. Measure ionic currents from squid axon –To determine contribution of Na+ and K+ Measure in normal saline with Na + and Na+- free saline

22. Ion currents underlying the AP Membrane Potential Ionic Currents Total current normal saline K+ current Na+ free saline outward inward

23. Ion currents underlying the AP Ionic Currents Na+ current outward inward Ionic Currents Total current normal saline K+ current Na+ free saline outward inward Subtract K+ current from total

24. Ion currents underlying the AP 1.The Na+ current activates quickly and then inactivates quickly 2.The K+ current activates more slowly and persists longer

25. Ionic Basis of Action Potentials 0 mV -80 mV P K >>P Na P Na >>P K P K >>P Na E Na EKEK Time 

26. Ion currents underlying the AP Membrane Potential Ionic Currents K+ current After-hyperpolarization P k(leak) + P k(volt) P k(leak)

27. The after hyperpolarization coincides –with the persistent K+ current and absent Na+ current

28. Stimulus & Threshold The stimulus depolarizes the membrane –Experimentally applied current –Synaptic potential –Receptor potential

29. Threshold The membrane potential at which Na flowing into the cell exactly equals the K flowing out of the cell A fraction more stimulus depolarization is required to ‘fire’ an action potential

30. Threshold Potential 0 mV -80 mV Small stimulus Below threshold Larger stimulus Reaches threshold

31. Membrane depolarization Increased Na permeability Na+ entry Positive Feedback The AP is regenerative and displays all-or-none behaviour

32. Why does the AP stop rising? 1.As Vm  E Na, Na+ inflow stops 2.Na+ channels inactivate 3.K+ channels open, K+ outflow starts E Na

33. Refractory Period 1.A second stimulus very soon after the first will not fire an AP (Absolute) 2.With a delay, a second stronger stimulus will cause a small AP (Relative) 3.With longer delay a second AP can be fired

34. Absolute refractory period Relative refractory period A B C

35. Why is there Refractory Period? The Na channel stays inactivated for a short period of time after it closes Inactivated Open Closed Active Closed Active

36. Summary & Key Concepts 1.The AP is controlled by rapid changes in ionic permeability 2.Permeability is a function of voltage- gated ion channels 3.Threshold potential 4.Positive feedback 5.Refractory period has two phases